Diabetic nephropathy (DN) is the leading cause of renal failure in Western societies. Development of new therapies is hindered by the lack of a clear understanding of disease pathogenesis. Recent studies revealed that loss of kidney podocytes is an early cardinal feature that contributes to the development and progression of DN; however, the underlying mechanism of podocyte loss remains unclear. Podocytes are specialized, terminally differentiated cells that maintain the kidney's filtration barrier. Recent studies have shown that hyperglycemia and advanced glycation end products (AGEs) increase oxidant stress leading to podocyte apoptosis. AGE-induced podocyte apoptosis is mediated by the activation of the FOXO4 transcription factor. The goal of this study is to study the role of FOXO4 and a protein deacetylase (SIRT1) that acts upstream of FOXO4 pathway in podocyte apoptosis of diabetes. Deacetylation of FOXO3 by SIRT1 enhances the expression of antioxidant enzymes and DNA damage repair genes, but decreases the expression of apoptosis genes. SIRT1 is repressed in diabetic rats and patients with DN. We hypothesize that SIRT1 repression in the diabetic milieu increases FOXO4 acetylation. Thus, the expression of FOXO4 target genes shifts from oxidant detoxification and DNA damage repair to apoptosis; and podocytes are apoptosis prone and oxidatively stressed in diabetes. The first aim of the project is to determine the role of the SIRT1/FOXO4 pathway in oxidant generation and apoptosis. The second aim is to confirm the in vitro findings in animal models of diabetes. The effects of hyperglycemia and AGEs on FOXO4 acetylation will be determined. The role of SIRT1 in hyperglycemia- and AGE-induced FOXO4 acetylation will be assessed by gene knockdown and over-expression of SIRT1. The effects of AGE and high glucose on oxidant stress will be quantified by flow-cytometric measurements of DCF-DA fluorescence and dimerization of homovanillic acid. Apoptosis will be assessed by flow cytometry, TUNEL staining, and caspase assays. Gene expression will be quantified by real-time PCR and Western blot. The role of SIRT1 repression by AGEs will be determined by benfotiamine treatment of db/db mice to reduce AGEs formation. The role of SIRT1 in apoptosis will be assessed in podocyte-specific SIRT1 knockout mice with streptozotocin-induced diabetes. The long-term objectives of the study are to elucidate the pathogenesis of podocyte loss in human and identify novel targets for treatment of DN.